A strain of Staphylococcus pyogenes (NIH-R01-GAS) was isolated from the blood of an 18-year-old patient with Jobs syndrome who had recently received various antibiotic treatments including levofloxacin. Susceptibility testing revealed the isolate was resistant to the fluoroquinolone antibiotics. Quinolone resistance-determining regions (QRDRs) of both the parC subunit of topoisomerase and DNA gyrase A are conserved in a number of bacteria belonging to different genera. We designed polymerase chain reaction (PCR) primers flanking the QRDRs of these two genes from S. pyogenes on the basis of the amino acid sequence homologies among S. pneumoniae, E. coli, and S. aureus. The amino acid sequences deduced from the amplified gene products from S. pyogenes demonstrated significantly high homology to the same regions of these two genes in S. pneumoniae. Compared with the quinolone-sensitive ATCC strain, the quinolone-resistant isolate of S. pyogenes presented point mutations in DNA gyrase A and in the parC subunit of topoisomerase IV. Recently, we acquired another clinical strain of S. pyogenes (NIH-R02-GAS) with increased MICs to some of the fluoroquinolone antibiotics. Amino acid sequences of ParC and GyrA of this isolate demonstrated point mutations only slightly different from those of the first isolate, with serine-79 of the ParC changed to alanine rather than tyrosine, and no mutation of serine-81 of GyrA. Mutation of methionine-99 to leucine was common to GyrA of both isolates. We propose that the minor change in point mutations in the gyrA and uncommon point mutation in the parC gene in strain NIH-R02-GAS may explain the striking differences in fluoroquinolone susceptibilities in these two isolates of S. pyogenes. Sequencing of other regions will be performed.